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Members of the Fusarium graminearum species complex are important cereal pathogens worldwide and belong to one of at least nine phylogenetically distinct species. We examined 298 strains of the F. graminearum species complex collected from wheat or barley in Japan to determine the species and trichothecene chemotype. Phylogenetic analyses and species-diagnostic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLPs) revealed the presence and differential distribution of F. graminearum sensu stricto (s. str.) and F. asiaticum in Japan. F. graminearum s. str. is predominant in the north, especially in the Hokkaido area, while F. asiaticum is predominant in southern regions. In the Tohoku area, these species co-occurred. Trichothecene chemotyping of all strains by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 (70%) out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by use of 15 PCR-RFLP markers developed in this study. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.

Fifty-eight isolates representing 39 Pythium species and 17 isolates representing nine Phytophthora species were chosen to investigate intra- and intergeneric relationships with sequence analysis of three genomic areas. The internal transcribed spacer regions (ITS1 and ITS2), including the 5.8S gene of the ribosomal DNA were PCR amplified with the universal primers ITS1 and ITS4. On the other hand 563 bp of the cytochrome oxidase II (cox II) gene was amplified with the primer pair FM66 and FM58 for Pythium and FM75 and FM78 for Phytophthora. The 658 bp partial beta-tubulin gene was amplified with the forward primer BT5 and reverse primer BT6. Maximum parsimony analysis of the three DNA regions revealed four major clades, reflective of sporangial morphology. Clade 1 was composed of Pythium isolates that bear filamentous to lobulate sporangia. Clade 2 represents Pythium isolates that bear globose to spherical zoosporangia or spherical hyphal swellings. Meanwhile Phytophthora isolates were lumped into Clade 3 wherein the papillate, semipapillate and nonpapillate species occupied separate subclades. Lastly, Clade 4 was composed of Pythium species that bear subglobose sporangia resembling the papillate sporangia observed in Phytophthora. Hence a number of species (Ph. undulata, P. helicoides, P. ostracodes, P. oedochilum and P. vexans) have been proposed to be the elusive intermediate species in the Pythium-to-Phytophthora evolutionary line.

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The sequences of ITS regions in 30 species and two groups of the genus Pythium were resolved. In the phylogenetic trees, the species were generally divided into two clusters, referred to here as the F and S groups. The species in the two groups correspond in terms of their sporangial morphology, with the F group being filamentous/Iobulate and the S group being spherical. Genetic divergence within the F group was lower than that within the S group. Other morphological characteristics such as oogonial structure and sexual nature appeared to be unrelated to the groupings in these trees. An alignment analysis revealed common sequences to all the species and arrangements specific to each F or S group. It was found that the ITS region was a good target in designing species-specific primers for the identification and detection of Pythium species. In the tree based on 5.8S rDNA sequences, oomycetes are distantly related to other fungi but separated from algae in Chromista. Key WordsChromista; internal transcribed spacer; phylogeny; Pythium; 5.8S rDNA.Pythium is a large genus of the class Oomycetes including more than 80 species (van der Plaats-Niterink, 1981 ), some of which are important plant pathogens with worldwide distribution. The taxonomy of this genus is mainly based on the morphology of reproductive structures such as the oogonium, oospore, antheridium, and sporangium. Since Matthews (1931) and Sideris (1932) proposed the first keys for Pythium spp., several keys have been published as new species have been described. Researchers providing systematic taxonomy in the genus are Middleton (1943), Waterhouse (1967), and van der Plaats-Niterink (1981). Hendrix and Papa (1974) introduced the concept of 'species complexes' in relation to taxonomy of the genus Pythium. This is an arrangement in which the species of Pythium are lumped into 15 species groups based on their morphological characteristics. In the taxonomic keys for Pythium, the size of each structure and common morphological characteristics are taken as criteria, including: (1) the presence of sexual reproductive structures -homothallic or heterothallic; (2) the sporangial morphology-spherical, filamentous or Iobulate; (3) the oogonial wall charactersmooth or ornamental; (4) the oospore character-plerotic or aplerotic; and (5) the antheridial charactermonoclinous or diclinous, However, each author regarded different characteristics as more important. For example, Middleton (1943) used the sporangial morphology at the first branch, whereas Waterhouse (1967) frequently utilized the reproductive structure size in the separaCorresponding author. E-mail: kageyama(~cc.gifu-u.ac.jp tion of species. Van der Plaats-Niterink (1981) regarded the presence of reproductive structures and the oogonial wall character as more important criteria. These differences in the interpretation of the taxonomic value of each character have resulted in a confusing taxonomic system for the Pythium species. It is therefore necessary to determine which morphological characteristic is the ...

Fusarium fujikuroi is a pathogenic fungus that infects rice. It produces several important mycotoxins, such as fumonisins. Fumonisin production has been detected in strains of maize, strawberry, and wheat, whereas it has not been detected in strains from rice seedlings infested with bakanae disease in Japan. We investigated the genetic relationships, pathogenicity, and resistance to a fungicide, thiophanate-methyl (TM), in 51 fumonisin-producing strains and 44 nonproducing strains. Phylogenetic analyses based on amplified fragment length polymorphism (AFLP) markers and two specific genes (a combined sequence of translation elongation factor 1α [TEF1α] and RNA polymerase II second-largest subunit [RPB2]) indicated differential clustering between the fumonisin-producing and -nonproducing strains. One of the AFLP markers, EATMCAY107, was specifically present in the fumonisin-producing strains. A specific single nucleotide polymorphism (SNP) between the fumonisin-producing and nonproducing strains was also detected in RPB2, in addition to an SNP previously found in TEF1α. Gibberellin production was higher in the nonproducing than in the producing strains according to an in vitro assay, and the nonproducing strains had the strongest pathogenicity with regard to rice seedlings. TM resistance was closely correlated with the cluster of fumonisin-nonproducing strains. The results indicate that intraspecific evolution in Japanese F. fujikuroi is associated with fumonisin production and pathogenicity. Two subgroups of Japanese F. fujikuroi, designated G group and F group, were distinguished based on phylogenetic differences and the high production of gibberellin and fumonisin, respectively. IMPORTANCE Fusarium fujikuroi is a pathogenic fungus that causes rice bakanae disease. Historically, this pathogen has been known as Fusarium moniliforme, along with many other species based on a broad species concept. Gibberellin, which is currently known as a plant hormone, is a virulence factor of F. fujikuroi. Fumonisin is a carcinogenic mycotoxin posing a serious threat to food and feed safety. Although it has been confirmed that F. fujikuroi produces gibberellin and fumonisin, production varies among strains, and individual production has been obscured by the traditional appellation of F. moniliforme, difficulties in species identification, and variation in the assays used to determine the production of these secondary metabolites. In this study, we discovered two phylogenetic subgroups associated with fumonisin and gibberellin production in Japanese F. fujikuroi.

We aimed to simultaneously detect two pathogens causing strawberry diseases, Phytophthora nicotianae and P. cactorum, by multiplex polymerase chain reaction (PCR), and to survey their occurrence in the main strawberry production areas of Japan. Due to the need to combine different primer pairs for multiplex PCR and the low specificity of published specific primers for P. nicotianae and P. cactorum, new species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions of ribosomal DNA and the ras-related protein gene Ypt1, respectively. Specificity of the designed primers was demonstrated using 68 isolates, including Phytophthora spp., Pythium spp., and other soilborne pathogens. Multiplex PCR discriminated between P. nicotianae and P. cactorum in DNA mixtures of mycelia of the two species. Moreover, both species were detected in artificially and naturally infested soils, indicating that these markers can be used in diagnosis of strawberry diseases. For investigation of the geographic distribution of the two pathogens in Japan, soil samples were collected in 89 strawberry fields from eight prefectures (Gifu, Saga, Nara, Tochigi, Chiba, Shizuoka, Yamanashi, and Hokkaido) of Japan. The method that was developed was successfully applied to survey P. nicotianae and P. cactorum, and distribution of the two pathogens in strawberry plantings in Japan was determined.

This study was initiated to understand whether differential biological control efficacy of Enterobacter cloacae on various plant species is due to differences in the ability of E. cloacae to inactivate the stimulatory activity of seed exudates to Pythium ultimum sporangium germination. In biological control assays, E. cloacae was effective in controlling Pythium damping-off when placed on the seeds of carrot, cotton, cucumber, lettuce, radish, tomato, and wheat but failed to protect corn and pea from damping-off. Seeds from plants such as corn and pea had high rates of exudation, whereas cotton and cucumber seeds had much lower rates of exudation. Patterns of seed exudation and the release of P. ultimum sporangium germination stimulants varied among the plants tested. Seed exudates of plants such as carrot, corn, lettuce, pea, radish, and wheat were generally more stimulatory to P. ultimum than were the exudates of cotton, cucumber, sunflower, and tomato. However, this was not directly related to the ability of E. cloacae to inactivate the stimulatory activity of the exudate and reduce P. ultimum sporangium germination. In the spermosphere, E. cloacae readily reduced the stimulatory activity of seed exudates from all plant species except corn and pea. Our data have shown that the inability of E. cloacae to protect corn and pea seeds from Pythium damping-off is directly related to its ability to inactivate the stimulatory activity of seed exudates. On all other plants tested, E. cloacae was effective in suppressing damping-off and inactivating the stimulatory activity of seed exudates.Pythium ultimum is a widespread and important oomycete plant pathogen causing seed decay, pre-and postemergence damping-off, and root rot in many plant species (8). The sporangium is a major survival structure in soil, even though oospores are produced (29,30). This is especially true for asexual strains of P. ultimum (10). Sporangia are exogenously dormant propagules that will germinate in response to stimulants from the seeds of host plants (14,15,17,19,28). This response occurs within the first few hours of seed germination, resulting in seed infection within the first 24 h after sowing (13). The molecules in seed exudates eliciting these rapid responses are largely unknown (14) but are believed to consist of long-chain unsaturated fatty acids (28).Enterobacter cloacae is a common plant-associated rhizobacterium effective in controlling Pythium diseases (3, 5-7, 9, 12, 13, 16, 22, 23, 31, 33, 34). Other biological control agents such as Pseudomonas and Bacillus species suppress Pythium diseases largely through the biosynthesis of antibiotics or other Pythium-inhibitory substances (2, 32, 34). However, no antibiotic production or parasitism has been found in E. cloacae, even though E. cloacae attaches to the hyphae of P. ultimumcolonizing seed surfaces (16). The control of Pythium dampingoff by E. cloacae can be attributed to its ability to reduce or eliminate responses of sporangia to germinating seeds by metabolizing long-chain...